Nitric oxide delivery devices

ABSTRACT

An example of a nitric oxide delivery device includes a medium, a working electrode in contact with the medium, and a reference/counter electrode in contact with the medium and electrically isolated from the working electrode. The medium includes a source of nitrite ions and a Cu(II)-ligand complex. A nitric oxide permeable material separates the medium from an external environment that is to contain blood.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.13/852,841, filed Mar. 28, 2013, which itself claims the benefit of U.S.Provisional Application Ser. No. 61/617,886, filed Mar. 30, 2012, bothof which are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under EB000783 andEB004527 awarded by the National Institutes of Health (NIH). TheGovernment has certain rights in the invention.

BACKGROUND

Nitric oxide (NO) has been shown to have several important physiologicalfunctions, including its unique vasodilating properties, cancer-fightingpotency, anti-platelet activity, and anti-microbial/anti-viral activity.In some instances, NO can be used to control infection, prevent biofilmformation, and minimize inflammation and fibrosis. Although NO is astable radical, it is highly reactive with hemoglobin and oxygen, thusmaking delivery of NO to the target site challenging. Stablehydrophilic, as well as hydrophobic NO donors may be employed to takeadvantage of the potency of NO for a wide range of biomedicalapplications. NO release polymeric materials and coatings based ondiazeniumdiolate chemistry have been used to inhibit platelet adhesion.While these materials and coatings do exhibit NO release, theinstability of diazeniumdiolates and other NO donors (e.g.,S-nitrosothiols) render the commercialization of these materials andcoatings challenging. For example,(Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]-diazen-1-ium-1,2-diolate(MAHMA/NO) dispersed in a silicone rubber matrix may, in some instances,prevent thrombus formation on the surface of intravascular sensors.MAHMA/NO may also greatly reduce platelet activity when employed withina polymer coating on the inner walls of extracorporeal circuits.However, MAHMA/NO and its corresponding diamine precursor tend to leachfrom the surface of the polymer matrix and back react with an oxidativeintermediate of NO to form potentially toxic nitrosamines.

BRIEF DESCRIPTION OF THE DRAWINGS

Features and advantages of examples of the present disclosure willbecome apparent by reference to the following detailed description anddrawings, in which like reference numerals correspond to similar, thoughperhaps not identical, components. For the sake of brevity, referencenumerals or features having a previously described function may or maynot be described in connection with other drawings in which they appear.

FIG. 1A is a schematic view of an example of a nitric oxide deliverydevice including a two-electrode configuration;

FIG. 1B is a schematic view of an example of the nitric oxide deliverydevice including a three-electrode configuration;

FIG. 2A is a schematic view of another example of the nitric oxidedelivery device in the form of a single lumen catheter;

FIG. 2B is a schematic, partially cross-sectional view of an example ofa nitric oxide delivery triple lumen catheter positioned in a bloodvessel;

FIG. 2C is an enlarged, schematic view of the chemical reaction takingplace at the working electrode in the catheter of FIG. 2B;

FIG. 2D is a schematic, perspective view of a dual lumen catheter havinga dedicated NO generating lumen that houses an example of a mediumincluding a source of nitrite ions and a Cu(II)-ligand complex;

FIG. 3A is a schematic view of another example of the nitric oxidedelivery device in the form of a planar patch;

FIG. 3B is an enlarged view of the inside of the planar patch of FIG.3A;

FIG. 4A is a schematic view of an example of a gas delivery systemincluding a nitric oxide generating system and a blood oxygenator;

FIG. 4B is a schematic view of another example of a gas delivery systemincluding a nitric oxide generating system and an inhalation unit;

FIG. 5A is a graph illustrating the nitric oxide flux from an example ofthe nitric oxide delivery device over a 24 hour period;

FIG. 5B is an expanded portion of the graph of FIG. 5A;

FIGS. 6A and 6B are graphs illustrating the effect of electrochemicalgeneration of nitric oxide from a catheter surface on biofilm formationfor E. Coli (FIG. 6A) and A. baumannii (FIG. 6B);

FIG. 7 is a graph illustrating the nitric oxide flux from anotherexample of the nitric oxide delivery device over a 14 hour period withthe device being turned on and off at different points;

FIGS. 8A and 8B are graphs illustrating the effect of nitric oxidemediated E. Coli biofilm dispersal in an example of the method using asource of nitrite ions;

FIG. 9 is a graph illustrating the change in potential detected from anexample of one type of the catheters disclosed herein, including thepotential change after two 2-hour intervals when the catheter was turnedon electrochemically to generate nitric oxide gas;

FIGS. 10A and 10B are respective representations of a photograph of anexample catheter that electrochemically generated nitric oxide in vivoand of a control catheter that did not electrochemically generate nitricoxide in vivo;

FIG. 11 is a cyclic voltammogram of a bulk solution including 1 mMCu(II)-tri(2-pyridylmethyl)amine (CuTPMA), a buffer, and varying levelsof nitrite;

FIG. 12 is a graph illustrating the modulation of NO generated in a bulksolution (including 4 mM CuTPMA, a buffer, and 100 mM nitrite) byapplying −0.2 V, −0.3 V, and −0.4 V (vs. 3 M Ag/AgCl);

FIGS. 13A through 13H are graphs illustrating NO release and flux from asingle lumen catheter over 8 days (D1=day 1 . . . D8=day 8) using acathodic voltage of −0.4 V (vs. 3 M Ag/AgCl), which was periodicallyturned off to examine the effect on NO generation;

FIG. 14 is a graph illustrating NO release and flux from a single lumencatheter over about 12 hours using varying cathodic voltages;

FIG. 15 is a graph illustrating the effect of NO release from single anddual lumen catheters on thrombus reduction;

FIG. 16A is a finite element analysis illustrating a simulated NOconcentration profile near a dual lumen catheter surface (partitioncoefficient K=S_(silicone)/S_(water)=7, D_(NO) (polymer)>D_(NO)(water));

FIG. 16B is a concentration plot across the line drawn through the duallumen catheter in FIG. 16A;

FIGS. 17A and 17B are graphs illustrating the effect of nitric oxidemediated E. Coli biofilm dispersal in an example of the method using amedium including a source of nitrite ions and a Cu(II)-ligand complex;

FIG. 18 is a cyclic voltammogram of a bulk solution including 1 mMCu(II)-tri(2-pyridylmethyl)amine (CuTPMA), a buffer, and varying levelsof nitrite and oxygen;

FIG. 19A shows the infrared spectra of N₂O produced using N₂O standardswith different levels of N₂O;

FIG. 19B is a calibration curve obtained by integration of the N₂Ofeature peaks at 2235 cm⁻¹ and 2212 cm⁻¹ in FIG. 19A; and

FIG. 19C shows the infrared spectra of N₂O produced using solutions witha fixed level of CuTPMA and different levels of nitrite.

DETAILED DESCRIPTION

The present disclosure relates generally to nitric oxide deliverydevices. In the example devices disclosed herein, the amount of NO thatis generated may be controlled in order to have a desired effect in aparticular application. As one example, periodic NO generation may beused for killing bacteria in applications where clotting is not an issue(e.g., with urinary catheters). As another example, a steadyphysiologically-relevant flux of NO for a predetermined time period maybe generated to reduce thrombus formation and prevent infection forintravascular catheter applications. As still another example, a steadyphysiologically-relevant flux of NO for a predetermined time period maybe generated in and delivered from a patch attached to the outside of anexternal wound to assist in wound healing.

Some examples of the nitric oxide delivery device disclosed hereinenable one to perform a pulsed electrochemical method within gaspermeable polymeric materials to generate and modulate the release ofnitric oxide (NO) through the gas permeable polymeric material. Thesepulsed electrochemical methods may also be used in oxygenators todeliver nitric oxide as well as oxygen to blood during extracorporealcirculation (ECC). In any of these examples, the nitric oxide iselectrochemically generated by the reduction of nitrite ions by Cu(I)ions, which are generated at the surface of a working electrode that ismade of a copper containing conductive material, or a base materialcoated with a copper containing conductive material. In some instances,the pulsed electrochemical method is triggered in response to a detectedchange in potential or pH (e.g., at the surface of a catheter). It hasalso been found that during NO generation of the pulsed electrochemicalmethod, the working electrode becomes passivated, for example, with anoxide or hydroxide layer, which can inhibit or deleteriously impact theability to generate of NO. These examples of the method disclosed hereininvolve a two-step applied potential sequence, where one step generatesCu(I) ions and thus NO, and the other step cleans and refreshes thepassivated working electrode surface. The cleaning and refreshing stepprepares the working electrode surface for subsequent NO generation.

As used herein, it is to be understood that a “copper containingconductive material” is any material that contains copper and is able torelease Cu(I) when an appropriate potential is applied. Examples ofthese materials include copper or copper alloys. The copper containingconductive material may be in the form of a wire, a mesh, an ink orpaint that is applied to a surface (e.g., on an inner surface of thehousing), copper nanoparticles that are incorporated/embedded into anelectrically conducting polymer matrix or a conductive carbon paste, orany other desirable form. One example of the copper containingconductive material is a copper wire material.

Other examples of the nitric oxide delivery device disclosed hereininvolve a Cu(II)-ligand complex that functions as a mediator. Theseexamples enable one to perform an electrochemical method that uses acathodic voltage alone to generate and modulate the release of NO. Inthese examples, the NO is electrochemically generated by reducing theCu(II)-ligand complex to a Cu(I)-ligand complex, the Cu(I) of which thenfunctions to reduce nitrite ions (NO₂ ⁻) to NO. The NO that is generatedis not bound to the reduced Cu(I) center of the ligand complex, and thusis capable of being transported out of the medium in which is itgenerated or permeated through to an external environment withoutperforming additional steps to oxidize the ligand complex. The ratio ofCu(I)-ligand complex to Cu(II)-ligand complex at the surface of an inertwire electrode can be controlled by controlling the applied potential.This enables one to control the amount of NO generated for a givenconcentration of nitrite and Cu(II)-ligand complex. In some instances,the cathodic voltage electrochemical method is triggered in response toa detected change in potential or pH (e.g., at the surface of acatheter).

Any of the examples of the nitric oxide delivery device disclosed hereinmay be a two electrode or a three electrode system. Some examples of thetwo electrode system are shown in FIGS. 1A, 2A, 2B, 2D, 3A, 3B, 4A, and4B; while an example of the three electrode system is shown in FIG. 1B.In the two electrode configurations, a working electrode and a referenceor counter electrode (referred to herein as a reference/counterelectrode) are used and current passes through the reference/counterelectrode. In the three electrode configurations, a working electrode, areference electrode, and a counter electrode are used. In these systems,the applied voltage is measured versus the reference electrode, but thecurrent passes through the counter electrode. A potentiostat may be usedto operate the circuit when either the two or the three electrode systemis used.

Referring now to FIGS. 1A and 1B, schemes of electrochemical generationof nitric oxide using the working electrode 12 in a two electrode system10 and a three electrode system 10′ are respectively depicted. In theseexample systems 10, 10′, the generated nitric oxide is transferred to anitric oxide analyzer NOA (e.g., a nitric oxide chemiluminescenceanalyzer) via an outlet 36 for quantitation of the amount of NOgenerated. It is to be understood however, that the two and threeelectrode systems 10, 10′ may not include the outlet 36 and the NOA(see, e.g., FIGS. 4A and 4B). In the examples shown in FIGS. 1A and 1B,the systems 10, 10′ also include a purge gas inlet 32 and an air or N₂gas inlet 34, which may be used to purge samples (e.g., a source ofnitrite ions 26 or a medium 27 including the Cu(II)-ligand complex andthe source of nitrite ions) during operation of the systems 10, 10′.

While these systems 10, 10′ may be used to generate NO using any of themethods disclosed herein, it is to be understood that the schemes ofelectrochemical generation illustrated in FIGS. 1A and 1B may beimplemented into other devices, such as catheters (e.g., single lumencatheters shown as reference numeral 100 in FIG. 2A and multi-lumencatheters shown as reference numeral 100′ in FIGS. 2B and 100″ in FIG.2D), patches (shown as reference numeral 200 in FIG. 3A), and gasdelivery systems (shown as reference numeral 300 in FIGS. 4A and 300′ inFIG. 4B). Rather than transferring the NO to the NO analyzer forquantitation, these devices 100, 100′, 100″, 200, 300, and 300′ eitherrelease the generated NO into the external/surrounding environmentthrough the walls of the catheter tubing or through the planar patchmaterial, or transport the generated NO in a gas stream to a bloodoxygenator (FIG. 4A) or an inhalation unit (FIG. 4B). In some thesedevices, the external environment may be exposed to or may containblood. In reference to FIGS. 1A and 1B, the schemes of electrochemicalgeneration will be described, and in reference to FIGS. 2A through 4B,various examples of the devices incorporating examples of these schemeswill be described.

Referring now to FIG. 1A, the two electrode system 10 includes theworking electrode 12 and the reference/counter electrode 14. In theexamples of the method in which the source of nitrite ions 26 is used,the working electrode 12 may be an electrode made of the coppercontaining conductive material or another electrode (e.g., platinum,gold, carbon, mercury, etc.) coated with the copper containingconductive material. In the examples of the method in which the medium27 is used, the working electrode 12 may be an electrode made ofplatinum, gold, carbon (e.g., glassy carbon) or a carbon coatedmaterial, mercury, etc. In any examples of the method disclosed herein,the reference/counter electrode 14 may be silver/silver chloride or someother reference electrode or pseudo reference electrode.

Conductive leads 16, 18 are respectively and electrically connected tothe working electrode 12 and the reference/counter electrode 14. Theconductive leads 16, 18 electrically connect the respective electrodes12, 14 to the electronics 20 (e.g., a potentiostat) that are used tocontrol the applied voltage, and in some instances, record potentialshift changes and compare potential shift changes to a preset thresholdvalue. Conductive leads 16, 18 may be made of any conductive material,examples of which include copper wires, platinum wires, stainless steelwires, aluminum wires, etc.

The working electrode 12 in FIG. 1A is shown in a glass tube 22, whichis configured so that an end 24 of the working electrode 12 is exposedto the source of nitrite ions 26 or the medium 27, but the source ofnitrite ions 26 or medium 27 does not enter the tube 22. Thereference/counter electrode 14 may also be contained within a glass tube22′. The tube 22′ is configured so that an end 25 of thereference/counter electrode 14 is exposed to the source of nitrite ions26 or to the medium 27, but the source of nitrite ions 26 or medium 27does not enter the tube 22′.

In an example, the source of nitrite ions 26 may be a water soluble,inorganic nitrite salt in an aqueous solution or within a hydrogel(e.g., hydroxymethylcellulose, poly(vinyl alcohol) (PVA), gelatin,etc.). Some examples of water soluble, inorganic nitrite salts includealkali metal and alkaline earth metal nitrite salts. Specific examplesinclude nitrite salts of Li, Na, K, Rb, Ca, and Mg. Most other metalsalts are also soluble in water, for example, Al salts and Fe salts. Onespecific example of the source of nitrite 26 is NaNO₂.

In another example, the source of nitrite ions 26 may be a lipophilicquaternary ammonium nitrite species soluble in an organic polymericphase. This particular source of nitrite ions 26 may not be suitable forthe examples of the catheter disclosed herein that have a modifiedwall/exterior surface. For the other examples that may include thelipophilic quaternary ammonium nitrite species, the lipophilicquaternary ammonium nitrite salt may be chosen from tetradodecylammoniumnitrite, tridodecylmethylammonium nitrite, tetradecylammonium nitrite,and tetraoctylammonium nitrite. The organic polymeric phase may bechosen from polyurethane, poly(vinyl chloride), polymethacrylate, andpolydimethylsiloxane (PDMS). The organic polymeric phase may be dopedwith a relatively high concentration of the lipophilic quaternaryammonium nitrite salt. The high concentration of the lipophilicquaternary ammonium nitrite salt will depend, at least in part, on thepolymer matrix used. In an example, the high concentration ranges fromabout 1 mM to about 300 mM. In one example when the lipophilicquaternary ammonium nitrite species is used as the source of nitriteions 26, the working electrode 12 (i.e., the source of Cu(I)) may benanoparticles of the copper containing conductive material embedded inthe organic polymer phase.

In examples that utilize the medium 27, any of the previously describedwater soluble, inorganic nitrite salts in aqueous solution or within thehydrogel may be used as the source of nitrite ions. In addition, themedium 27 includes the Cu(II)-ligand complex, which is water soluble.Examples of the Cu(II)-ligand complex includeCu(II)-tri(2-pyridylmethyl)amine (CuTPMA),Cu(II)-tri(2-dimethylamino)ethyl]amine (CuMe₆Tren),Cu(II)-tri(2-pyridylmethyl)phosphine (CuTPMP), and combinations thereof.These structures are shown below:

While some examples of the Cu(II)-ligand complex are provided herein, itis to be understood that other water soluble Cu(II)-complexes may beused.

The source of nitrite ions 26 or the medium 27 may also include a bufferand/or another additive that aids in driving the reduction reaction ofCu(I) with nitrite. Examples of suitable buffers are phosphate bufferedsaline (PBS) or 3-(N-morpholino)propanesulfonic acid (MOPS). An exampleof the other suitable additive is ethylenediaminetetraacetic acid(EDTA). EDTA helps drive the reduction reaction of Cu(I) with nitrite(Cu(I)+NO₂ ⁻+2H⁺→Cu(II)+NO+H₂O) to the product side by chelating withCu(II) stronger than with Cu(I). As an example, the source of nitriteions 26 includes 1 M NaNO₂, 25 mM EDTA and 0.138 M NaCl in 1 M PBS (pH7.2). As another example, the medium 27 includes 4 mM CuTPMA, 0.4 MNaNO₂, and 0.2 M NaCl in 0.5 M MOPS buffer (pH 7.2). When a Ag/AgClreference is utilized, a fixed level of chloride ions is provided (e.g.,as NaCl) in the source of nitrite ions 26 or the medium 27 so that thereference electrode can maintain a constant potential (EMF).

Electrochemical generation of nitric oxide using the two electrodesystem 10 of FIG. 1A involves application of voltage (e.g., continuouslyor as pulses) to the working electrode 12, where current passes throughthe reference/counter electrode 14.

In examples using the source of nitrite ions 26 (i.e., not the medium 27including the Cu(II)-ligand complex), a cathodic voltage pulse isapplied to the working electrode 12 to clean the surface (including theend 24) of the working electrode 12. In these examples, the cathodicpulse may be applied for a period of time that is suitable forrefreshing the working electrode 12 surface. In an example, the time forwhich the cathodic pulse is applied to refresh the working electrode 12ranges from about 1 second to about 10 minutes. In these examples, ananodic voltage pulse is then applied to the working electrode 12. Theanodic voltage pulse produces a low concentration of Cu(I) ions at theelectrode 12 surface. The Cu(I) ions produced at the end 24 reactdirectly with nitrite in the source of nitrite ions 26 to generatenitric oxide gas. The anodic voltage pulse is applied to the workingelectrode 12 for a limited time interval ranging from about 1 second toabout 10 minutes, at least in part because Cu₂O/CuOH forms on theelectrode 12 when the anodic voltage pulse is applied. The oxide formedon the surface of the working electrode 12 in these examples of themethod interferes with the generation of nitric oxide gas. When it isdesired to generate NO again, the cathodic pulse is applied to clean theelectrode 12, and then the anodic pulse is applied to generate NO. Toobserve consistent reduction of nitrite to NO in these examples of themethod, the two-step potential sequence (i.e., cathodic pulse followedby anodic pulse) is continuously and repeatedly applied to the workingelectrode 12.

It is to be understood that the time frames provided for application ofthe cathodic voltage pulse and the anodic voltage pulse are examples andmay be varied depending upon the amount of NO to be generated and/or thetime needed to refresh the working electrode 12 surface. Generally, ifless Cu(I) is generated during the anodic step, than the cathodic stepwill be shorter because less time is needed to refresh the workingelectrode surface. As an example, it is believed that a cycle thatincludes a 5 second cathodic pulse and a 5 second anodic pulse may beused. In this example, the NO production may be relatively low due, atleast in part, to rapid depletion of nitrite near the working electrode12. Nitrite arrives at the electrode 12 surface by diffusion, and therate of NO production may be faster than the rate of nitrite diffusion.However, such low levels of NO may be desirable in some instances.

In examples using the medium 27 including both the source of nitriteions and the Cu(II)-ligand complex, a cathodic voltage is applied to theworking electrode 12. The cathodic voltage may be applied continuously,in pulses (e.g., more negative voltage followed by less negativevoltage), or using a desirable on/off sequence. When applied, thecathodic voltage pulse produces a low concentration of the Cu(I)-ligandcomplex at the electrode 12 surface. The Cu(I)-ligand complex producedat the end 24 reacts directly with nitrite in the medium 27 to generatenitric oxide gas. The cathodic voltage may be applied to the workingelectrode 12 for any time interval up to, for example, 30 days. In someinstances, it is believed that the voltage may be applied continuouslyfor even longer than 30 days. When it is desired to stop generating NO,the cathodic voltage is no longer applied to the electrode 12. In someexamples, the cathodic voltage may be turned on periodically (e.g., for1 to 3 hours per day to kill bacteria), and the usable life of thedevice may be for multiple months. In other examples, when the voltageis continuously applied, the voltage may be modulated to be more or lessnegative in order to increase or decrease, respectively, the rate of NOproduction, and thus the flux of NO emitted from the surfaces of thedevice (e.g., catheter, wound healing patch, etc.). In some example, amore negative voltage may be applied for a shorter time than a lessnegative voltage in order to conserve the nitrite source in the solution27 for a longer period. Variations on the application of the cathodicvoltage in this example of the method are also contemplated as beingwithin the purview of this disclosure.

Referring now to FIG. 1B, the three electrode system 10′ includes theworking electrode 12, the reference electrode 28, and the counterelectrode 30. Similar to the two electrode system 10, the workingelectrode 12 that is used with the source of nitrite ions 26 may be anelectrode made of the copper containing conductive material or anotherelectrode (e.g., platinum, gold, carbon, mercury, etc.) coated with thecopper containing conductive material, and the working electrode 12 thatis used with the medium 27 may be an electrode made of platinum, gold,carbon (e.g., glassy carbon) or a carbon coated material, mercury, etc.In an example of the three electrode system 10′, the reference electrode28 is silver/silver chloride and the counter electrode 30 is platinum(e.g., a platinum mesh). In the three electrode system 10′, thereference electrode 28 may also be an ion-selective pseudo-referenceelectrode (e.g., a sodium-selective electrode or a potassium-selectiveelectrode).

Conductive leads 16, 18 are respectively and electrically connected tothe working electrode 12 and the counter electrode 30. A conductive lead38 electrically connects the reference electrode 28 to the working andcounter electrodes 12, 30. The conductive leads 16, 18, 38 electricallyconnect the respective electrodes 12, 30, 28 to the electronics 20(e.g., a potentiostat) that are used to control the applied voltages,and in some instances, record potential shift changes and comparepotential shift changes to a preset threshold value. The conductiveleads 16, 18, 38 may be made of any of the conductive materialspreviously described.

In FIG. 1B, the working electrode 12 is set up in a similar manner tothat described in reference to FIG. 1A. Also in FIG. 1B, the counterelectrode 30 is set up in a similar manner to that described for thereference/counter electrode 14 in reference to FIG. 1A. The counterelectrode 30 used in the three electrode system 10′ may be inserted intothe source of nitrite ions 26 or the medium 27. Electrochemicalgeneration of nitric oxide using the three electrode system 10′ of FIG.1B involves application of voltage (e.g., continuously or as pulses) tothe working electrode 12, where current passes through the counterelectrode 30. The voltage applied to the working electrode 12 andthrough the counter electrode 30 is measured against the referenceelectrode 28. This system 10′ may be used to apply the previouslydescribed two-step potential sequence (i.e., cathodic pulse followed byanodic pulse) to the working electrode 12 in the presence of the sourceof nitrite ions 26 to generate NO, or may be used to apply thepreviously described cathodic potential to the electrode 12 in thepresence of the medium 27 to generate NO.

Some of the components of the systems 10, 10′ of FIGS. 1A and 1B may beincorporated into medical devices, such as catheters 100 (shown in FIG.2A), 100′ (shown in FIG. 2B), or 100″ (shown in FIG. 2D), or planarpatches 200 (shown in FIGS. 3A and 3B), or gas delivery devices 300(shown in FIG. 4A) or 300′ (shown in FIG. 4B). These devices 100, 100′,100″, 200, 300, 300′ may be operated to selectively generate NO asdescribed above. In some examples, the catheters 100, 100′, 100″ may beconfigured to detect the presence of bacteria (e.g., biofilm) on itssurface, and in response, turn on the electrochemical cell to generateNO to disperse the attached bacteria and keep the surface free ofbacteria. As will be described further below, in these examples, thehousing 40 of the catheter 100, 100′, 100″ is ion conductive so that thesurface potential of the housing 40 can be measured and an externalreference electrode is utilized.

In the devices 100, 100′, 100″, 200, 300, 300′, the electronics thatapply the desired potentials, and in some instances record and comparepotential shifts and values, may be minimized to be secured to thenitric oxide permeable material (e.g., housing 40) or to the nitricoxide generating system (shown in FIGS. 4A and 4B), and may be operatedvia a battery or another source of energy (e.g., solar generator, anenergy harvesting device, etc.). Furthermore, while the followingdescription relates to examples of the two electrode system, it is to beunderstood that the three electrode system may also be implemented intothe medical devices.

Referring now to FIG. 2A, the single lumen catheter 100 illustrated is atwo electrode system (similar to system 10) that includes a housing 40that is permeable to nitric oxide. It is to be understood that thesingle lumen catheter 100 may be particularly desirable for applicationsthat do not require blood sampling, drug and/or nutrient infusion, etc.For example, the single lumen catheter 100 may be particularly suitablefor studies demonstrating the effectiveness of the electrochemical NOgenerating methods disclosed herein. For many medical applicationshowever, blood sampling and/or drug and/or nutrient infusion isdesirable, and thus the multi-lumen catheters 100′, 100″ describedhereinbelow may be more desirable for those types of applications.

Still referring to FIG. 2A, the housing 40 keeps the source of nitriteions 26 or the medium 27 and the electrodes 12, 14 separated from theexternal environment. The housing material is selected so as to preventthe source of nitrite ions 26 or the medium 27 from leaking out of thehousing 40 while allowing NO gas to permeate through the housing 40 tothe external environment. Examples of suitable materials for the housing40 include silicone rubber, biomedical grade polyurethane,polytetrafluoroethylene (PTFE), polytetrafluoroethylene derivatives(e.g., ethylene tetrafluoroethylene (ETFE), fluorinatedethylene-propylene (FEP), perfluoroalkoxy (PFA), etc.),polymethacrylate, and poly(vinyl chloride). The housing 40 used in thecatheter 100 may have a cylindrical geometry.

Both the working electrode 12 and the reference/counter electrode 14 arepositioned within the housing 40. The electrodes 12, 14 are positionedsuch that they are electrically isolated from one another. The exposedelectrodes 12, 14 may be physically separated from one another. As shownin FIG. 2A, in an example, a portion of the reference/counter electrode14 that is adjacent to the working electrode 12 within the housing 40may be coated with an insulating layer 42 in order to ensure electricalisolation of the two electrodes 12, 14. This insulating layer 42 isdescribed further hereinbelow.

Conductive leads 16, 18 are electrically connected to the respectiveelectrodes 12, 14. The leads 16, 18 may be part of the respectiveelectrodes 12, 14, or may be separate wires that are electricallyconnected to the respective electrodes 12, 14. The leads 16, 18 extendfrom the electrodes 12, 14 (which are inside the housing 40) toelectronics (not shown) which are outside of the housing 40. FIG. 2Aillustrates an example in which the leads 16, 18 are part of therespective electrodes 12, 14. In this example, the working electrode 12includes the coiled portion within the housing 40, and the lead portion16 extending outside of the housing 40. Also in this example, thereference/counter electrode 14 includes the coiled portion within thehousing 40, and the lead portion 18 that is partially inside of thehousing and partially outside of the housing 40. In examples in whichthe leads 16, 18 are separate wires that are connected to the electrodes12, 14, it is to be understood that the leads 16, 18 may be the samematerial as, or different materials than the electrodes 12, 14.

The housing 40 may be sealed at areas where the leads 16, 18 passtherethrough. In an example, the housing 40 is sealed using a siliconerubber seal/sealant (an example of which shown in FIG. 2B as referencenumeral 43).

The housing 40 also contains the source of nitrite ions 26 or the medium27. Any of the sources 26 or media 27 previously described may be usedin this example device 100. The source of nitrite ions 26 or the medium27 is contained within the housing 40 such that the working electrode 12and the reference/counter electrode 14 are in contact with the source ofnitrite ions 26 or the medium 27.

While not shown in FIG. 2A, it is to be understood that when the sourceof nitrite ions 26 is used, the working electrode 12 may be formed of anink or paint that includes the copper containing conductive material,and the reference/counter electrode 14 may be formed of an ink or paintthat includes silver or another suitable conductive material. In thisexample, the electrodes 12, 14 may be screen printed or otherwise formedon the inner walls of the housing 40.

As briefly mentioned above, areas or portions of one or more of theelectrodes 12, 14 and/or the leads 16, 18 may be coated with aninsulating layer 42. As illustrated in FIG. 2A, a portion of thereference/counter electrode 14 that is adjacent to the working electrode12 within the housing 40 may be coated with the insulating layer 42. Inthis example, at least a portion of the reference/counter electrode 14within the housing 40 (e.g., the coiled portion shown in FIG. 2A)remains exposed. Also as illustrated in FIG. 2A, the leads 16, 18 may becoated with the insulating layer 42. In these examples, ends of theleads 16, 18 may remain exposed to electrically connect to suitableelectronics. Any insulating material may be used for the insulatinglayer 42, examples of which include polytetrafluoroethylene (PTFE),polyethylene, silicone, etc. In other examples, the electrodes 12, 14and/or the leads 16, 18 may remain uncoated.

Electrochemical generation of nitric oxide using the catheter 100 ofFIG. 2A and the source of nitrite ions 26 involves application of acathodic voltage pulse to the working electrode 12 to clean the surfaceof the working electrode 12, and then application of an anodic voltagepulse to the working electrode 12 to produce a concentration of Cu(I)ions at the electrode 12 surface. The Cu(I) ions produced at the surfacereact directly with nitrite in the source of nitrite ions 26 to generatenitric oxide gas, which permeates through the housing 40 to the externalenvironment.

Electrochemical generation of nitric oxide using the catheter 100 ofFIG. 2A and the medium 27 involves application of a cathodic voltage tothe working electrode 12 to produce a concentration of Cu(I)-ligandcomplex in the medium 27, which react directly with nitrite in themedium 27 to generate nitric oxide gas, which permeates through thehousing 40 to the external environment.

As previously mentioned, in either examples of the method disclosedherein, the potential applied to the working electrode 12 may becontrolled to turn NO release on or off. An advantage of this control isthat nitrite salts may be dissolved in high concentrations andincorporated into reservoirs in the lumen of the catheter 100. As anexample involving a dual lumen biomedical catheter, one lumen may beused as a nitrite salt reservoir, and the second lumen may be used tosample blood, infuse therapeutic agents, etc. It is believed that a 150μm thick layer of a 1 M nitrite solution can produce a 1×10⁻¹⁰mol·min⁻¹·cm⁻² NO flux continuously for at least 100 days in acylindrical arrangement (e.g., a catheter) or planar arrangement (e.g.,the patch described below).

It has been found (see Example 15) that using high concentrations of thenitrite salts in the source 26 or the medium 27 significantly reducesthe amount of N₂O that may be generated during the electrochemicalmethods disclosed herein. In the examples disclosed herein, at least 100mM nitrite is used as the source of nitrite ions 26 or in the medium 27,and amounts lower than 100 mM cannot be used. These levels of nitritesuppress the formation of N₂O to negligible levels. As an example, asource of nitrite ions 26 or a medium 27 including 400 mM nitrite mayresult in less than 5% N₂O in the total gas species that is generatedusing the methods disclosed herein. It is believed that the excessnitrite competitively binds to the Cu center of the ligand complex(after the mediated reduction of nitrite to NO by the complex) so thatthe electrogenerated NO leaves the copper-ligand complex, rather thansuch a complex being reduced electrochemically again in the presence ofanother nitrite ion to form N₂O. This prevents the formation ofsignificant levels of N₂O.

It is to be understood that while illustrated as a single lumen catheterconfiguration in FIG. 2A, in other examples (e.g., when to be used inclinical practice and other medical applications), the electrochemicalgenerating concepts disclosed herein may be implemented using amulti-lumen catheter. The multi-lumen catheter 100′ (FIG. 2B) or 100″(FIG. 2D) may include two or more lumens (three, four, etc.), as long asone of the lumens is dedicated for NO generation. The dedicated NOgenerating lumen of the multi-lumen catheters may be configured forperforming any of the methods disclosed herein. One example of thededicated NO generating lumen of the multi-lumen catheter may includethe medium 27 and the electrodes 12, 14, and the application of acathodic pulse may generate NO. Another example of the dedicated NOgenerating lumen of the multi-lumen catheter may include the source ofnitrite ions 26 and the electrodes 12, 14, and the application of thetwo-step potential sequence (i.e., cathodic pulse followed by anodicpulse) may clean the electrode 12 and generate NO.

An example of a multi-lumen catheter 100″ having at least one lumen L₁dedicated for NO generation is depicted in FIG. 2D. While this exampleis shown with the medium 27, it is to be understood that the electrodes12, 14 may be selected to be used with the source of nitrite ions 26 andthe pulsed electrochemical method disclosed herein.

In the example of FIG. 2D, the lumen L₁ includes the working electrode12 and the reference/counter electrode 14 so that at least some of theelectrode 12, 14 is in contact with the medium 27 contained within thelumen L₁. While not shown, it is to be understood that the lumen L₁ mayalso be sealed, for example, with a silicone rubber cap.

Within the lumen L₁, when a cathodic voltage is applied (e.g., −0.4 Vvs. reference electrode 14), the Cu(II)-ligand complex in the medium 27is reduced to the Cu(I)-ligand complex. The Cu(I)-ligand complex reactsdirectly with nitrite in the solution 27 to generate nitric oxide gas,which permeates through the housing 40 to the external environment. Thereactions (i.e., (Cu(II)-ligand complex→Cu(I)-ligand complex andCu(I)-ligand complex+NO₂ ⁻+2H⁺→Cu(II)-ligand complex+NO+H₂O) takingplace within the solution are shown in FIG. 2D.

In the multi-lumen catheter 100″ example, there is at least one openlumen L₂ in addition to the lumen L₁ dedicated for NO generation. Bloodcan be removed through the open lumen L₂ of the catheter 100″, ortherapeutic solutions can be infused into a patient through the openlumen(s) L₂.

In any of the catheter 100, 100′, 100″ examples disclosed herein, thewall, and thus the exterior surface S, of the housing 40 may be modifiedto detect any charged species (e.g., negatively charged or positivelycharged bacteria) that is sitting on the exterior surface S. Bacteria,and in particular biofilm, that forms on the exterior surface S of thecatheter 100, 100′, 100″ may contribute to catheter related infections.The catheter 100, 100′, 100″ having the modified surface can detect thebacteria, and in response, can initiate NO generation from within thehousing 40. The catheter 100, 100′, 100″ can release NO that isgenerated by the electrochemical methods disclosed herein. The releasedNO acts as an effective antimicrobial and biofilm dispersal agent. Thereleased NO may also exhibit potent antithrombotic activity.

The wall and exterior surface S of the housing 40 may be modified withany additive(s) that will render the surface S with the ability todetect charge on the surface S. In an example, the additive(s) areimpregnated into the exterior surface S. It is to be understood that anyof the housing materials mentioned herein may be modified with acation/anionic salt and a plasticizer. Examples of the cation/anionicsalt include tetradodecylammonium tetrakis(4-chlorophenyl)borate,tridodecylmethylammonium tetrakis(bis-trifluormethylphenyl borate), etc.Examples of the plasticizer include dibutyl sebacate, dioctyl sebacate,nitrophenyloctyl ether, etc. In an example, silicone tubing isimpregnated with tetradodecylammonium tetrakis(4-chlorophenyl)borate anddioctyl sebacate in an m-xylene solution.

In an example, the modified exterior surface S is capable of detectingcharged species that are in contact with the surface S by measuring thevoltage between the inner solution/hydrogel and the outer contact phase(e.g., the blood, using an external reference electrode). Theelectronics operatively connected to the catheter 100, 100′, 100″ areable to record negative and positive potential shifts, which depend uponthe number of bacteria on the surface S of the catheter 100, 100′, 100″.The potential shifts are measured from an initial operating potential ofthe catheter 100, 100′, 100″ (i.e., a background potential). In general,more bacteria present on the surface S results in a greater negative orpositive potential shift from the background potential. Storage/memoryassociated with the electronics may be programmed with a threshold valueof potential shift. The threshold value of potential shift may be basedupon an undesired number of bacteria present on the surface S. Forexample, it may be determined that a 100 mV shift in potential is anindication of a critical mass of bacteria on the surface S, and thisvalue may be stored as the threshold value to which recorded values arecompared. When the amount of bacteria present on the surface S reachesthe undesired number, the recorded potential shift will exceed thethreshold value. In an example, the potential shifts are recorded by ahigh impedance voltmeter.

The electronics (which may include a controller running computerreadable code stored on a non-transitory, computer readable medium) arealso configured to recognize that the threshold value of potential shifthas been reached or exceeded, and in response, will automaticallytrigger electrochemical NO production. In other words, upon recognizingthat the recorded potential shift meets or exceeds the threshold value,the electronics will initiate the electrochemical pulse method disclosedherein (when the source of nitrite ions 26 is used) or the constantcathodic voltage electrochemical method disclosed herein (when themedium 27 is used).

In an example in which the source of nitrite ions 26 is used, theelectronics are configured to first apply a cathodic voltage pulse tothe working electrode 12 to clean the surface of the electrode 12, andthen apply an anodic voltage pulse to the working electrode 12 toproduce a low concentration of Cu(I) ions at the working electrode 12surface. The Cu(I) ions produced at the surface react directly withnitrite in the source of nitrite ions 26 to generate nitric oxide gas,which permeates through the housing 40 to the external environment. Thereleased NO disperses the bacteria on the surface S. As a result ofbacteria dispersal, the surface potential shifts back to the backgroundvalue at which the catheter 100, 100′, 100″ was operating initially.Upon recognizing that that background potential is again reached, theelectronics are configured to turn off NO generation (i.e., stop theelectrochemical pulse method). The catheter 100, 100′, 100″ thenoperates at the background potential until the accumulation of bacteriaon the surface S is enough to cause another potential shift.

In an example in which the medium 27 is used, the electronics areconfigured to first apply a cathodic voltage to the working electrode 12to reduce the Cu(II)-ligand complex to produce a low concentration ofCu(I)-ligand complex. The Cu(I)-ligand complex then reacts directly withnitrite in the medium 27 to generate nitric oxide gas, which permeatesthrough the housing 40 to the external environment. The released NOdisperses the bacteria on the surface S. As a result of bacteriadispersal, the surface potential shifts back to the background value atwhich the catheter 100, 100′, 100″ was operating initially. Uponrecognizing that that background potential is again reached, theelectronics are configured to turn off NO generation (i.e., stop theapplication of the cathodic voltage). The catheter 100 or 100′ thenoperates at the background potential until the accumulation of bacteriaon the surface S is enough to cause another potential shift.

FIG. 2B schematically illustrates an example of a triple lumen catheter100′ positioned within a blood vessel 44 (also showing red blood cells46). In this example, lumens L₁ and L₃ are open lumens used for samplingblood and/or infusing therapeutic agents (represented by the verticalarrows at the open ends of the lumens L₁ and L₃. Lumen L₂ is thededicated NO generating lumen. In this example, the lumen L₂ includesthe source of nitrite ions 26 and the electrode 12 made of or coatedwith the copper containing conductive material. It is to be understoodthe lumen L₂ may also be configured with the medium 27 and itsassociated electrode 12. While not shown in FIG. 2B, this example couldalso include an external reference electrode that would be in contactwith the sample phase (e.g., the blood in the blood vessel 44), ifdetection of adhered bacteria 48 on the surface S of the device 100′ viaeither a surface charge effect or local pH change were desired to signalthe initiation of electrochemical NO release.

In this example, bacteria 48 on the surface S has been detected and apotential shift resulting from the bacteria 48 has been recognized asexceeding the pre-programmed threshold value. As such, electrochemicalpulses have been initiated using electrodes 12, 14, and NO is generated.The reaction (i.e., (Cu(I)+NO₂ ⁻+2H⁺→Cu(II)+NO+H₂O) taking place at theworking electrode 12 is shown in FIG. 2C. The generated NO permeatesthrough the housing 40 into the surrounding environment, where itcontributes to the prevention of smooth muscle cell proliferation, theprevention of platelet activation/thrombosis, and kills bacteria 48.

In still another example, the housing 40 may be modified to detectlocalized pH changes at the exterior surface S. The housing wall andexterior surface S may be doped with a chemical (e.g., a pH sensitivematerial) that allows electrochemical detection of the pH changes. In anexample, the housing 40 can be doped with any proton ionophore, such astridodecylamine (TDDA), along with from about 10 mol % to about 50 mol %(relative to the ionophore) of any tetraphenylborate species (lipophilicanion site).

The pH sensitive catheter may be operated in combination with thepreviously mentioned reference electrode external to the housing 40. Theexternal reference electrode facilitates monitoring of the potentialacross the pH sensitive housing 40, where the potential is a function ofthe pH of the external environment (including bacteria or other cellsadhering to the external surface S) being monitored. As such, thepotential between the reference/counter electrode 14 and the externalreference electrode tracks the pH of the external environment. In anexample, a potential change due to any difference in proton activity atthe exterior surface S of the housing 40 (measured using the externalreference electrode in contact with a solution outside of the housing40) versus proton activity at the inner surface e (i.e., pH buffer inthe source of nitrite ions 26 or the medium 27, measured usingreference/counter electrode 14) may be detected using a high impedancevoltmeter. This potential change indicates the presence of bacteria orother cells adhering to the exterior surface S of the catheter housing40. A given pH change as determined from the measured potential willthen trigger the electrochemical NO generation process, as describedabove when using surface charge to detect the presence of cells.

Referring now to FIGS. 3A and 3B, the planar patch 200 and an explodedview of its interior are respectively depicted. The planar patch 200illustrated is a two electrode system (similar to system 10) thatincludes the housing 40 that is permeable to nitric oxide. The housing40 of the planar patch 200 functions in the same manner as the housing40 used for the catheter 100, 100′. 100″. In this example however, thehousing 40 has the shape of a relatively flat cube, relatively flatrectangular box, or other relatively flat three-dimensional shape. Thehousing 40 does have a length, width, and a depth; however the depth maybe relatively small so that the planar patch 200 is flexible and able toconform to the shape of a desired subject (e.g., a limb, appendage, etc.that the planar patch is affixed, adhered, or otherwise secured to). Theplanar patch 200 may also be constructed so that only the surface thatis to be adjacent to the desired subject is made of the permeablematerial, while the remainder of the surfaces is impermeable to NO. Inthis example then, NO is releasable from the desired surface alone.

In this example, the working electrode 12 is a mesh 12′, which may be anetwork of wires or screen printed lines. The material of the workingelectrode 12, 12′ may vary depending upon whether the source of nitriteions 26 or the solution 27 is used. Similarly, the reference/counterelectrode 14 is a metal-containing mesh 14′, which may be a network ofmetal (Pt) or other conductive material (Ag/AgCl) wires or screenprinted lines. In an example of the planar patch 200 in which the sourceof nitrite ions 26 is used, the working electrode 12 may also be formedof nanoparticles of the copper containing conductive material dissolvedin a suitable polymer matrix.

The meshes 12′, 14′ (or, in an example, the mesh 14′ and the polymermatrix containing copper containing nanoparticles) are electricallyisolated from one another by a separator 44. Examples of suitableseparators 44 include polymeric materials, such as polyethylene,polypropylene, polytetrafluoroethylene, poly(vinyl chloride), or otherlike materials. While the separator 44 is electrically insulating, it isalso capable of conducting ions (i.e., is ionically conducting).

Conductive leads 16, 18 are electrically connected to the respectiveelectrode meshes 12′, 14′. In this example, the leads are separate wiresthat are electrically connected to the respective meshes 12′, 14′. Theleads 16, 18 extend from the meshes 12′, 14′ (which are inside thehousing 40) to electronics (not shown) which are outside of the housing40.

The housing 40 also contains the source of nitrite ions 26 or the medium27. Any of the sources previously described may be used in this exampledevice 200. The source of nitrite ions 26 or the medium 27 is containedwithin the housing 40 such that at least the working electrode mesh 12′is in contact with the source of nitrite ions 26 or the medium 27.

In an example in which the source of nitrite ions 26 is used,electrochemical generation of nitric oxide using the planar patch 200 ofFIG. 3 involves application of a cathodic voltage pulse to the workingelectrode mesh 12′ to clean the surface of the mesh 12′, and thenapplication of an anodic voltage pulse to the working electrode mesh 12′to produce a low concentration of Cu(I) ions at the mesh surface. TheCu(I) ions produced at the surface react directly with nitrite in thesource of nitrite ions 26 to generate nitric oxide gas, which permeatesthrough the housing 40 to the external environment. To observeconsistent reduction of nitrite to NO, the two-step potential sequence(i.e., cathodic pulse followed by anodic pulse) is continuously andrepeatedly applied to the working electrode mesh 12′.

In an example in which the medium 27 is used, electrochemical generationof nitric oxide using the planar patch 200 of FIG. 3 involvesapplication of a cathodic voltage to the working electrode mesh 12′ toproduce a low concentration of Cu(I)-ligand complex which reactsdirectly with nitrite in the solution 27 to generate nitric oxide gas,which permeates through the housing 40 to the external environment.

Referring now to FIG. 4A, an example of a gas delivery device 300 isdepicted. This gas delivery device 300 includes a nitric oxidegenerating system 10 similar to that previously described in referenceto FIG. 1A (except there is no outlet to an NO analyzer). It is to beunderstood that system 10′ shown in FIG. 1B may also be used in thesystem 300. The nitric oxide in the system 10 may be generated using anyof the methods disclosed herein utilizing the source of nitrite ions 26or the medium 27.

The gas delivery device 300 also includes an inlet conduit 49 fordelivering oxygen gas (O₂) to the source of nitrite ions 26 or medium 27in contact with the electrodes 12, 14. The inlet conduit 49 may be anysuitable polymeric or other tubing attached to an oxygen gas generator(not shown).

The oxygen gas stream that is introduced into the system 10 picks up thenitric oxide that is generated in the source of nitrite ions 26 or themedium 27 as a result of the electrochemical method(s) disclosed herein.The resulting stream of oxygen gas and nitric oxide is then transportedout of the system 10 through an outlet conduit 52. It is to beunderstood that this gas stream may include some contaminants. Theoutlet conduit 52 may be a tube that has low or no permeability to atleast the oxygen gas and the nitric oxide. The length of the outletconduit 52 may also be relatively short in order to avoid loss of gasbefore the stream is delivered to the oxygenator 51.

The stream is transported as a result of pressure from the oxygen gasgenerator (e.g., a compressed gas cylinder with a regulator to controlthe flow rate).

The outlet conduit 52 is configured to transport the stream of oxygengas and nitric oxide (O₂+NO or NO+O₂) from the system 10 to anoxygenator 51, which includes membrane(s) 50 capable of filtering andcleaning the gas stream. In this particular example, the oxygenator 51is a blood oxygenator, which includes a housing 53 with a blood inlet, ablood outlet, and a gas inlet.

The gas inlet of the housing 53 is operatively connected to the outletconduit 52. More particularly, the gas inlet directs the stream ofoxygen gas and nitric oxide from the outlet conduit 52 into membranes 50that are contained within the housing 53. In this example, each membrane50 is a hollow polymeric fiber having a first or interior surface I anda second or exterior surface E. A single blood oxygenator housing 53 mayinclude thousands of hollow polymeric fibers. The stream of oxygen gasand nitric oxide is introduced adjacent to the first or interior surfaceI. The walls of the hollow polymer fibers act as filters, allowing onlythe oxygen gas and the nitric oxide from the stream to permeatetherethrough (while trapping contaminants therein). As such, the cleanedstream of oxygen gas and nitric oxide exits from the second or exteriorsurface E into any blood contained within the housing 53 (as shown inthe expanded portion of FIG. 4A).

The NO in this example serves to locally prevent platelet adhesion andactivation on the second or exterior surface E of the membranes 50. Theeffect of the NO is localized since it reacts immediately withoxyhemoglobin to form met-hemoglobin. When the blood exits theoxygenator 51, NO is no longer present in the cleaned stream. As such,the blood, containing a cleaned stream of oxygen gas, can then exit thehousing 53 and be delivered to a patient.

While one example of the gas delivery system 300 is depicted, it is tobe understood that various other configurations may be utilized, forexample, the blood oxygenator 51 may have a different design.

Referring now to FIG. 4B, another example of a gas delivery device 300′is depicted. This gas delivery device 300′ also includes the nitricoxide generating system 10 similar to that previously described inreference to FIG. 1A (except there is no outlet to an NO analyzer). Itis to be understood that system 10′ shown in FIG. 1B may also be used inthe system 300′. The nitric oxide in the system 10 may be generatedusing any of the methods disclosed herein utilizing the source ofnitrite ions 26 or the medium 27.

Similar to the gas delivery device 300, this device 300′ also includesthe inlet conduit 49 for delivering oxygen gas (O₂) to the source ofnitrite ions 26 or medium 27 in contact with the electrodes 12, 14, andthe outlet conduit 52. As described above, the oxygen gas stream that isintroduced into the system 10 picks up the nitric oxide that isgenerated in the source of nitrite ions 26 or the medium 27 as a resultof the electrochemical method(s) disclosed herein. The resulting streamof oxygen gas and nitric oxide is then transported out of the system 10through the outlet conduit 52 as discussed above.

The outlet conduit 52 is configured to transport the stream of oxygengas and nitric oxide (O₂+NO or NO+O₂) from the system 10 to anoxygenator 51′, which includes a housing 53′ and a membrane(s) 50′capable of filtering and cleaning the gas stream. In this particularexample, the oxygenator 51 is part of an inhalation unit 56, which alsoincludes a patient delivery system 54 to deliver the cleaned stream ofoxygen gas and nitric oxide to a patient for the purpose of inhalationtherapy.

A gas inlet of the housing 53′ is operatively connected to the outletconduit 52. More particularly, the gas inlet directs the stream ofoxygen gas and nitric oxide from the outlet conduit 52 towards themembrane 50′ that divides the housing 53′. In this example, the membrane50 is a polymeric sheet having a first surface I and a second surface E.The stream of oxygen gas and nitric oxide is introduced adjacent to thefirst surface I. The membrane 50′ acts as a filter, allowing only theoxygen gas and the nitric oxide from the stream to permeate therethrough(while trapping contaminants in the one side 57 of the housing 53′). Thecleaned stream of oxygen gas and nitric oxide exits from the secondsurface E.

In this example, the cleaned stream of oxygen gas and nitric oxide isdelivered to a patient (e.g., for inhalation) through the patientdelivery system 54 (which may include a tube and a respirator).

While one example of the gas delivery system 300′ is depicted, it is tobe understood that various other configurations may be utilized. Forexample, a single polymer tube may form the inlet conduit 49 and theoutlet conduit 52, and is positioned within the source 26 or medium 27where NO is generated. In this example, the polymer tube would bepermeable to the NO, and the stream of oxygen gas or air transportedthrough the tube would pick up the NO through the permeable tube (i.e.,NO would diffuse through the polymer tubing and join the gas stream).The polymer tube could be configured to deliver the stream (includingoxygen gas and nitric oxide) directly to the patient. The polymer tubemay include a membrane 51′ so that contaminants are not delivered to thepatient.

The NO generation methods described herein may be controlled toselectively generate NO at a desired time. For example, control of thepotential applied to the working electrode 12 can turn on the generationof NO or can turn off the generation of NO. For efficient bactericidalactivity, it may be desirable to turn NO generation on and off at leastonce a day, where the on cycle ranges from about 1 hour to about 2 hoursor about 3 hours. In some instances, it may also be desirable to turn NOgeneration on and off multiple times within a day. In other examples,the device may be configured to sense the presence and/or absence ofbacteria and, in response, turn on and/or off NO generation,respectively. The flux of NO that is generated may also be modulated byaltering an amount of a surface area of the working electrode 12 or mesh12′ that is exposed to the source of nitrite ions 26 or the medium 27(e.g., changing the length of the electrode 12 or mesh 12′), by alteringa concentration of nitrite in the source of nitrite ions 26 or byaltering a concentration of nitrite and/or Cu(II)-ligand complex in themedium 27, by altering a magnitude of the cathodic voltage (e.g., a morenegative voltage for a short time followed by a less negative voltagefor a longer time) and/or the anodic voltage, by altering the pH, and/orby altering the concentration of additive(s) that are included.

In the examples disclosed herein, the NO generation may take place inair.

To further illustrate the present disclosure, examples are given herein.It is to be understood that these examples are provided for illustrativepurposes and are not to be construed as limiting the scope of thepresent disclosure.

EXAMPLES Example 1

A silicone rubber tubing (2 cm length, 0.51 mm inner diameter, and 0.94mm outer diameter) was sealed at one end with silicone rubber sealant. A2 cm long polytetrafluoroethylene (PTFE)-coated silver/silver chloridewire was used as the reference electrode, and a 2 cm long PTFE-coatedcopper wire was used as the working electrode. The bare copper wire hadan outer diameter of 0.127 mm, and coated copper wire had an outerdiameter of 0.152 mm. The PTFE was removed from the ends of thesilver/silver chloride wire and the copper wire in 20 mm and 10 mmlengths, respectively. The exposed ends of the respective wires werecoiled separately. The coiled ends were inserted into the siliconerubber tubing so that the silver/silver chloride wire and the copperwire were not in direct metallic connection.

A source of nitrite was loaded into the tubing. The source of nitriteincluded 1 M NaNO₂, 0.138 M NaCl, 0.02 M EDTA in 1 M phosphate bufferedsaline (PBS). The pH was adjusted to 6.8 with NaOH or using appropriateratios of phosphate salts.

The silicone rubber tubing was then sealed to form a catheter.PTFE-coated silver/silver chloride wire and PTFE-coated copper wireextended out of the silicone rubber tubing as respective leads to thereference and working electrodes.

A cathodic voltage (−0.7 V vs. NHE) was applied to the working electrodefor about 3 minutes, and then an anodic voltage (+0.2 V vs. NHE) wasapplied to the working electrode for about 3 minutes. The pulse sequencewas initiated for 1 hour every 2 hours over a 24 hour period. The NOflux from the surface of the silicone rubber tubing is shown in FIG. 5A.One 2 hour segment of FIG. 5A is shown expanded in FIG. 5B. The lefthand side of the graph illustrates when the voltage cycle is turned off,and the right hand side of the graph illustrates when the voltage cycleis turned on.

As illustrated in FIG. 5B, a catheter with a 2 cm long thin copperelectrode was able to generate an average NO flux of 0.6×10⁻¹⁰mol·cm⁻²·min⁻¹ when the previously described voltage cycle wasimplemented. In the absence of any voltage, nitric oxide was notgenerated and thus the NO flux was zero. It is noted that the averageflux reported in this Example is based upon the surface area of theentire silicon rubber tubing, not the copper electrode alone. Theresults shown in FIG. 5A illustrate that the potential applied to thecopper coated electrode can be controlled to turn on or off the releaseof NO through the walls of the silicone rubber tubing. The siliconerubber tubing provided a barrier against the leaching of nitrite fromthe catheter, but also allowed for the electrochemically generated NOgas to readily diffuse out of the catheter.

Example 2

A bacterial biofilm prevention study was performed using the NOgenerating catheters of Example 1. Two strains of bacteria were used,namely E. coli and A. baumannii.

The experiment was performed over a 1 week period with continuous flowof media using a Center for Disease Control (CDC) bioreactor. 4 NOgenerating catheters were immersed in the media containing either E.coli or A. baumannii. Two of the catheters were connected to anamperometric work station and a continuous potential program was appliedto the respective working electrodes. The potential program included 3minutes at −0.97 V vs. Ag/AgCl (−0.7 V vs. normal hydrogen electrode)for reduction, and −0.07 V vs. Ag/AgCl (+0.2 V vs. normal hydrogenelectrode) for liberation of NO. The other two catheters were used ascontrols. These control catheters contained the same nitrite saltsolution and wires, but were not linked to potentiostats.

At the end of the one week period, the catheters were stained withfluorescent dyes (SYTO-9 and propidium iodide) for 20 minutes in thedark. The catheters were put on a glass slide before being observed witha fluorescence microscope equipped with Fluorescent Illumination System(X-cite 120, EXFO) and appropriate filter sets. FIGS. 6A and 6Billustrate the bacterium counting results after 7 days. These resultsare based upon the staining and fluorescence imaging discussed above.The bacterium counting results show that the NO generating cathetersthat released NO had 90% less viable E. coli on their surfaces, and had98% less viable A. baumannii on their surfaces, when compared to thecontrol catheters (which did not release NO).

Example 3

A silicone rubber tubing (2 cm length, 0.51 mm inner diameter, and 0.94mm outer diameter) was sealed at one end with silicone rubber sealant. A2 cm long polytetrafluoroethylene (PTFE)-coated silver/silver chloridewire was used as the reference electrode, and a 2 cm long PTFE-coatedcopper wire was used as the working electrode. The bare copper wire hadan outer diameter of 0.127 mm, and coated copper wire had an outerdiameter of 0.152 mm. The PTFE was removed from the ends of thesilver/silver chloride wire and the copper wire in 20 mm and 10 mmlengths, respectively. The exposed ends of the respective wires werecoiled separately. The coiled ends were inserted into the siliconerubber tubing so that the silver/silver chloride wire and the copperwire were not in direct metallic connection.

A source of nitrite was loaded into the tubing. The source of nitriteincluded 1 M NaNO₂, 0.138 M NaCl, 25 mM EDTA in 1 M phosphate bufferedsaline (PBS). The pH was adjusted to 7.2 with NaOH or using appropriateratios of phosphate salts.

The silicone rubber tubing was then sealed to form a catheter.PTFE-coated silver/silver chloride wire and PTFE-coated copper wireextended out of the silicone rubber tubing as respective leads to thereference and working electrodes.

A cathodic voltage (−1.2 V vs. NHE) was applied to the working electrodefor about 30 seconds, and then an anodic voltage (+0.2 V vs. NHE) wasapplied to the working electrode for about 30 seconds. The pulsesequence was performed over a 14 hour period. The pulse sequence wasinitiated for about 2 hours at hours 0, 7 and 12, and for about 5 hoursfrom hour 3 to hour 8. The NO flux from the surface of the siliconerubber tubing is shown in FIG. 7. The results shown in FIG. 7 illustratethat the potential applied to the copper coated electrode can becontrolled to turn on or off the release of NO through the walls of thesilicone rubber tubing. The silicone rubber tubing provides a barrieragainst the leaching of nitrite from the catheter, but also allows forthe electrochemically generated NO gas to readily diffuse out of thecatheter.

Example 4

A bacterial biofilm dispersal study was performed using four NOgenerating catheters and one strain of bacteria, namely E. coli.

For each catheter, a silicone rubber tubing (2 cm length, 0.51 mm innerdiameter, and 0.94 mm outer diameter) was sealed at one end withsilicone rubber sealant. A 2 cm long polytetrafluoroethylene(PTFE)-coated silver/silver chloride wire was used as the referenceelectrode, and a 2 cm long PTFE-coated copper wire was used as theworking electrode. The bare silver/silver chloride wire had an outerdiameter of 0.125 mm, and the coated silver/silver chloride wire had anouter diameter of 0.176 mm. The bare copper wire had an outer diameterof 0.127 mm, and the coated copper wire had an outer diameter of 0.152mm. The PTFE was removed from the ends of the silver/silver chloridewire and the copper wire in 20 mm lengths to expose the bare electrodes.The exposed ends of the respective wires were coiled separately. Thecoiled ends were inserted into the silicone rubber tubing so that thesilver/silver chloride wire and the copper wire were not in directmetallic connection.

A source of nitrite was loaded into the tubing. The source of nitriteincluded 1 M NaNO₂, 0.138 M NaCl, 0.02 M EDTA in 1 M phosphate bufferedsaline (PBS). The pH was adjusted to 6.8 with NaOH or using appropriateratios of phosphate salts.

The silicone rubber tubing was then sealed to form the catheter.PTFE-coated silver/silver chloride wire and PTFE-coated copper wireextended out of the silicone rubber tubing as respective leads to thereference and working electrodes.

The experiment was performed over a 2 day period with continuous flow ofmedia (80 mL/hr) using a drip-flow bioreactor. The catheters wereimmersed in the media containing E. coli. Two of the catheters wereconnected to a potentiostat and a pulse sequence was applied to therespective working electrodes for 3 hours after two days in the flowingmedia with the cells without generating NO. These catheters are referredto as the NO releasing catheters. One cycle of the pulse sequenceincluded 30 seconds at −1.2 V vs. Ag/AgCl wire, and 30 seconds at +0.2 Vvs. Ag/AgCl wire. These NO releasing catheters were able to generate anaverage NO flux of 1.2*10⁻¹⁰ mol/cm²*min when this pulse sequence wasapplied. The other two catheters were used as controls. These controlcatheters contained the same nitrite salt solution and wires, but werenot connected to potentiostats.

At the end of the 2 day period, one NO generating catheter and onecontrol catheter were used for detection of viable bacteria on thecatheter surfaces by a plate counting method. The other NO generatingand control catheters were used for imaging. In particular, these twocatheters were stained with fluorescent dyes (SYTO-9 and propidiumiodide) for 20 minutes in the dark. The catheters were put on a glassslide before being observed with a fluorescence microscope equipped withFluorescent Illumination System (X-cite 120, EXFO) and appropriatefilter sets.

FIG. 8A illustrates the results observed for the one control catheterand the one NO releasing catheter obtained by the fluorescence imagingof total bacteria on the respective surfaces. As shown in FIG. 8A, thefluorescent imaging experiments indicate a dramatic reduction in numberof cells left on the surface for the NO releasing catheter.

FIG. 8B illustrates the bacterium counting results (Colony FormingUnits, CFU) after 2 days for the other control catheter and the other NOreleasing catheter. The bacterium counting results (FIG. 8B) illustratethat NO generating catheters that were turned on for only 3 hours of NOrelease had nearly a 3 log unit reduction in the number of living E.coli cells on their surface compared to the control.

Both the fluorescence imaging and the bacterium counting resultsdemonstrate the effectiveness of using the electrochemically modulatedNO release catheter to disperse bacteria biofilm after they have formedon the surface. In particular, the results indicate the effectivenessusing a one-time application of the NO release cycle for a relativelyshort period of time.

Example 5

A surface of a silicone rubber tubing (0.541 μm inner diameter, and 940μm outer diameter) was modified by impregnating the tubing withtetradodecylammonium tetrakis(4-chlorophenyl)borate (ETH 500) anddioctyl sebacate (DOS) in an m-xylene solution. Impregnation was allowedto take place for 24 hours, and then the tubing was air-dried overnightinside of a fume-hood. The dried tubing was then put in an oven of 120°C. for about 1 hour.

A source of nitrite was loaded into the tubing. The source of nitriteincluded 1 M NaNO₂, 0.138 M NaCl, 20 mM EDTA in 1 M phosphate bufferedsaline (PBS).

Two cm long of 127 nm PTFE-coated copper and PTFE-coated Ag/AgCl wireswere then inserted inside the tube containing the source of nitrite. Thetube was sealed at the open end with a silicone sealant and was curedovernight at room temperature. A portion of each of the wires extendedout of the silicone rubber tubing as respective leads to the referenceand working electrodes.

The control catheter was the same type of surface modified catheter withthe same source of nitrite and the same electrodes inserted inside thetubing, but the NO release was never electrochemically initiated.

The catheter and control catheter put inside respective drift flowbioreactors to grow biofilm on the respective surfaces. A standard ordouble junction commercial Ag/AgCl reference electrode was also placedinside the same channel with the catheter, and with the controlcatheter. 10% live bacteria was then passed through the respectivechannels. The background open circuit potential (BOCP) was measured for30 minutes vs. the respective external Ag/AgCl reference electrode. Thechambers were then inoculated with E. Coli bacteria for one hour andthen the media was flowed through continuously again. The open circuitpotential (OCP) measurement against the external Ag/AgCl referenceelectrode was recorded every hour.

For the sample including the catheter, when the change of 170 mV in OCPwas observed from baseline (see FIG. 9), an electrochemical pulse wasturned on to release NO for 2 hours. The electrochemical pulse used was−1.2 V for 30 seconds and +0.2 V for 30 seconds. The NO flux used forthe first NO release was 1×10⁻¹⁰ moles/cm²·min. The OCP was measuredagain after stopping the electrochemical pulse.

The OCP was again measured every hour until the next event (i.e., achange in OCP) was observed. This indicated that bacteria were againstarting to stick on the catheter surface. The electrochemical pulse wasagain turned on to release NO for 2 hours. The NO flux used for thesecond NO release was 0.5×10⁻¹⁰ moles/cm²·min. The electrochemical pulseused was −1.2V for 30 seconds and +0.1 for 30 seconds.

After the experiment, the catheter and the control catheter were takenout of the respective chambers and the surface bacteria were dispersedinto respective solutions via a homogenizer. The solutions were platecounted to determine the number of live bacteria left on the cathetersurface and the control catheter surface. The number of bacteria afterthe experiment with the catheter was found to be 36% less than thecontrol.

Example 6

Five example catheters (S1-S5) and four control catheters (C1-C4) wereprepared to assess the in vivo effects of the pulsed electrochemicalrelease of NO on thrombus formation. All of the example catheters andthe control catheters were prepared as previously described in Example3, except an additional length (about 3 inches) of inert tubing wasattached to the top end to enable insertion of the distal tips intorabbit veins.

White rabbits (2.5-3.5 kg, Myrtle's Rabbitry, Thompson's Station, Tenn.)were used for the thrombus experiment. Intramuscular injections of 5mg/kg xylazine injectable (AnaSed Lloyd Laboratories Shenandoah, Iowa)and 30 mg/kg ketamine hydrochloride (Hospira, Inc. Lake Forest, Ill.)were used to induce anesthesia before each rabbit experiment.Maintenance anesthesia was administered via a diluted intravenous (IV)infusion of ketamine (2 mg/ml) at a rate of 1.53 mg/kg/h. IV fluids ofLactated Ringer's were given at a rate of 33 ml/kg/h to maintain bloodpressure stability.

Example catheters S1, S3, S4 and S5 and control catheters C1-C4 wereimplanted in rabbit jugular veins and allowed to remain in the veins for6 hours. Example catheter S2 was implanted into a rabbit leg vein andwas allowed to remain for 6 hours. For each of the Example catheters, anelectrochemical pulse sequence was turned on, with the NO flux rangingfrom 0.8*10⁻¹⁰ moles/cm²·min to 1.1*10⁻¹⁰ moles/cm²·min to release NO.The particular NO flux at 37° C. for the respective Example catheters isshown in Table 1. For the control catheters, the electrochemical pulsesequence was turned off, with the NO flux <<0.3*10⁻¹⁰ moles/cm²·min.

After the 6 hours, the example and control catheters were explanted andthrombus on each example and control catheter was recorded via digitalphotography. Red pixels were counted from the photo using Image Jsoftware. The surface coverage was calculated using the red pixel data,and these results are shown in Table 1.

TABLE 1 Throm- Sample bus or Area/ Total Surface Control NO flux @ 37°C. cm² Area/cm² Coverage S1 ~1.1 * 10⁻¹⁰ moles/cm² · min 0.021 0.315 6.7% S2 ~1.0 * 10⁻¹⁰ moles/cm² · min 0.010 0.202  5.0% S3 ~1.1 * 10⁻¹⁰moles/cm² · min 0.039 0.202 19.3% S4 ~0.8 * 10⁻¹⁰ moles/cm² · min 0.0780.167 46.7% S5 ~0.9 * 10⁻¹⁰ moles/cm² · min 0.083 0.252 46.7% AVG.22.12%  C1 N/A 0.214 0.380 56.3% C2 N/A 0.263 0.360 73.1% C3 N/A 0.6901.107 62.4% C4 N/A 0.165 0.273 60.2% AVG. 63.0%As illustrated, each of the example catheters had less thrombusformation than each of the control catheters. For those examplecatheters when the NO flux was lower, there was a bit more thrombusformation. Overall, these results illustrate that in vivoelectrochemical generation of NO reduces thrombus formation.

A similar experiment was performed using an example catheter and acontrol catheter (as previously described), except that both of thecatheters were allowed to remain in the jugular veins for 8 hours. Anelectrochemical pulse sequence was turned on using the example catheter,but was not turned on for the control catheter. Upon extraction of theexample catheter and the control catheter from the rabbit veins, digitalphotographs were taken. These photographs are schematically representedin FIGS. 10A and 10B. As depicted, the example catheter used toelectrochemically generate NO in vivo had very little thrombusformation, as opposed to the control catheter, which was almostcompletely covered with thrombus.

Example 7

A three electrode system similar to that shown in FIG. 1B was used inthis example. A 0.0314 cm² gold disc electrode was used as the workingelectrode, a platinum coil was used as the counter electrode, and asilver/silver chloride electrode was used as the reference electrode.The bulk solution included 1 mM CuTPMA in 0.1 M MOPS buffer (pH 7.2)with different levels of nitrite in N₂ (i.e., 0 mM nitrite, 1 mMnitrite, 10 mM nitrite, and 100 mM nitrite).

Cyclic voltammetry (CV) was performed with a scan rate of 50 mV/s, andthe results are shown in FIG. 11. The reversible peaks in the absence ofnitrite correspond to a one electron reduction from Cu(II) to Cu(I), andthe characteristic catalytic peak in the presence of nitrite indicatesthe nitrite is catalytically reduced.

Similar CV experiments were performed with a 0.0314 cm² Pt discelectrode and a 0.0707 cm² glassy carbon disc electrode. The bulksolutions in these experiments included 1 mM CuTPMA in 0.1 M MOPS buffer(pH 7.2) with different levels of nitrite in N₂ (i.e., 0 mM nitrite, 10mM nitrite, and 100 mM nitrite). While not shown, the CV for each ofthese experiments was similar to the CV shown in FIG. 11.

Example 8

A three electrode system similar to that shown in FIG. 1B was used inthis example. A 0.071 cm² in surface area glassy carbon electrode wasused as the working electrode, a platinum coil was used as the counterelectrode, and a silver/silver chloride electrode was used as thereference electrode. The bulk solution included 4 mM CuTPMA and 100 mMnitrite in 0.1 M MOPS buffer (pH 7.2).

A cathodic voltage was applied to the bulk solution and was modulatedover time. As shown in FIG. 12, low, medium, and high flux of constantNO release can be modulated by applying −0.2 V, −0.3 V, and −0.4Vrespectively (vs. 3M Ag/AgCl reference electrode) in the bulk solution.In this example, the NO formed was electrochemically detected by achemiluminescence nitric oxide analyzer (NOA).

Example 9

A single lumen silicone rubber tubing (7.5 cm length, inner diameter1.47 cm, and outer diameter 1.96 cm) was sealed at one end with siliconerubber sealant. A polytetrafluoroethylene (PTFE)-coated silver/silverchloride wire (with 0.039 cm² surface area exposed) was used as thereference electrode, and a PTFE-coated platinum wire (with 0.079 cm²surface area exposed) was used as the working electrode. The exposedends of the respective wires were coiled separately. The coiled endswere inserted into the single lumen silicone rubber tubing so that thesilver/silver chloride wire and the copper wire were not in directmetallic connection.

A solution was loaded into the single lumen silicone rubber tubing. Thesolution included 2 mM CuTPMA, 0.4 M NaNO₂, and 0.2 M NaCl in 0.5 M MOPSbuffer (pH 7.2).

The silicone rubber tubing was then sealed to form a catheter.PTFE-coated silver/silver chloride wire and PTFE-coated copper wireextended out of the silicone rubber tubing as respective leads to thereference and working electrodes.

A dual lumen catheter was also prepared in a similar manner byintroducing the solution and electrodes into one of the two lumens(where the dedicated NO generating lumen is slightly larger than theopen lumen for blood of infusing agents).

A −0.4 V vs. Ag/AgCl voltage was applied to the platinum electrode ofthe single lumen catheter for 8 days, although there were some instancesat which the voltage was turned off. When the voltage was turned off atdays 1, 2, 5, 6, and 7, the NO generation significantly decreased. Theresults are shown in FIGS. 13A though 13H. As illustrated over thesefigures, the NO flux can be modulated by turning the voltage on and off,but when on, a relatively constant NO flux can be achieved.

Example 10

A single lumen silicone rubber tubing (7.5 cm length, inner diameter1.47 cm, and outer diameter 1.96 cm) was sealed at one end with siliconerubber sealant. A polytetrafluoroethylene (PTFE)-coated silver/silverchloride wire (with 0.039 cm² surface area exposed) was used as thereference electrode, and a PTFE-coated platinum wire (with 0.080 cm²surface area exposed) was used as the working electrode. The exposedends of the respective wires were coiled separately. The coiled endswere inserted into the single lumen silicone rubber tubing so that thesilver/silver chloride wire and the copper wire were not in directmetallic connection.

A solution was loaded into the single lumen silicone rubber tubing. Thesolution included 4 mM CuTPMA, 0.4 M NaNO₂, and 0.2 M NaCl in 0.5 M MOPSbuffer (pH 7.2).

The silicone rubber tubing was then sealed to form a catheter.PTFE-coated silver/silver chloride wire and PTFE-coated copper wireextended out of the silicone rubber tubing as respective leads to thereference and working electrodes.

A dual lumen catheter may be formed in a similar manner by introducingthe solution and electrode into one of the two lumens.

The cathodic voltage (vs. 0.2 M Cl⁻Ag/AgCl) was applied to the platinumelectrode of the single lumen catheter for about 12 hours. The voltagewas modified over this time period to illustrate the effect on themodulation of NO flux. The results are shown in FIG. 14. As illustrated,the NO flux can be modulated by applying different voltages, and in thisparticular example, the flux can vary from about 0.05 to about3.25×10⁻¹⁰ mol min⁻¹ cm⁻².

Example 11

The single and dual lumen catheters of Example 9 were used in 7 hour invivo testing to determine the efficacy of examples of the methoddisclosed herein using the medium 27.

Two of the single or dual lumen catheters were placed in rabbit jugularveins (n=3 rabbits) with one of the catheters “turned on” (−0.5 V) andthe other “turned-off” (not linked to potentiostat, i.e., the control).The degree of thrombus was assessed by imaging the catheters afterremoval. Red pixels were counted from the photo using Image J software.The surface coverage was calculated using the red pixel data. Theresults are shown in FIG. 15. The NO release catheters consistentlyexhibited reduced thrombosis, with an average 89% reduction in thrombusarea for the single lumen catheters when compared with the controlcatheters. The in vivo thrombosis experiments for the dual lumencatheters showed that the NO release catheters had an average reductionof 69% in thrombus area when compared with the control catheters.

The dual lumen catheters were asymmetric (where the dedicated NOgenerating lumen is slightly larger than the open lumen for blood ofinfusing agents). Although the asymmetry could have caused an unevendistribution of NO at the outer and inner surfaces of the lumens, thesilicone rubber material had a very high NO solubility and mobility.This material provided a reservoir for the generated NO and the improveddistribution of the gas. This was confirmed by finite element analysis(COMOSOL Multiphysics), as similar NO concentration was found near thesurfaces of the two respective lumens (see FIGS. 16A and 17B).

FIG. 16A shows the finite element analysis simulation results after 2hours of electrochemically generating NO from electrodes placed in theright side lumen. The grey scale code shows the concentration of NO atdifferent locations within the catheter lumens, within the polymerwalls, and within the adjacent solution. The highest concentrations arewithin the walls of the tubing, due, at least in part, to the highpartition coefficient for NO to solubilize in the silicone rubbermaterial. This high solubility enables there to be a relatively lowasymmetry in the concentrations of NO that exist in the solution phaseon both sides of the dual lumen catheter. FIG. 16B shows the actualrelative concentrations going from left to right across the width of thecatheter (i.e., across the line shown through the dual lumen catheter inFIG. 16A), starting in solution phase on the left side of the catheter.The highest levels are within the walls of the tubing, with the highestwithin the thin silicone wall between the two lumens of the catheter. Itis noted in FIG. 16B, that the concentration values on the Y-axis aremultiple by 10⁻⁵ (as shown at the top of the Y-axis).

Example 12

To assess the antimicrobial activity of the dual lumen catheters ofExample 9, the amount of surface-adhered bacteria was determined after ahighly inoculated media solution containing the microbes as flowedcontinuously over the surface of the catheters for several days. Thedual lumen catheters were tested in a drip flow system, which mimickedthe catheter environment in vivo. E. coli were grown on the catheterswith continuous nutrient flowing for 3 days and the NO release wasturned on for only 3 hours each day with a flux of 0.6×10⁻¹⁰ mol min⁻¹cm⁻².

The results are shown in FIGS. 17A and 17 B. Even with the relativelylow amount of periodic NO release, the plate counts showed a more than1000-fold decrease of viable bacteria on the channel surface in whichthe NO releasing catheters (n=5) were placed. The reduction of biofilmformation on the channel walls was so great that it could even beobserved visually without a microscope. In addition, more than 100-folddecrease in viable bacteria was observed on the catheter surfaces withNO release turned on periodically.

Example 13

When the medium 27 is used in some of the examples disclosed herein, acompeting reaction of oxygen with reduced Cu(I)TPMA may take place. Theeffect of oxygen was tested in this example. Cyclic voltammetry (CV) wasperformed with a scan rate of 50 mV/s using a bulk solution including 1mM CuTPMA in 0.1 M MOPS buffer (pH 7.2) with different levels of nitritein air (i.e., 0 mM nitrite, 1 mM nitrite, 10 mM nitrite, and 100 mMnitrite). A 2 mm gold disc working electrode and a Ag/AgCl referenceelectrode were used.

The results are shown in FIG. 18. The CV is similar in the presence andabsence of oxygen, suggesting no significant effect (i.e., does notsignificantly suppress NO production).

Example 14

It is believed that the Cu(II)-ligand complex cannot transport throughPDMS to any significant degree. A 7 day copper leaching test wasperformed, and the results confirmed this belief Catheters of Example 9were placed in PBS buffer at room temperature for 7 days. No copper wasdetected in the soaking solution by ICP-OES.

Example 15

The effect of nitrite level in the initial medium 27 on N₂O productionwas tested.

A calibration method was used for quantification of N₂O. First, standardN₂O solutions (i.e., 0.25 mM, 1 mM, 2 mM, and 2.5 mM N₂O) were preparedby adding different amounts of saturated N₂O solution to an airtightglass containing 10 mL of MOPS buffer which had been deaerated bypurging Ar for 30 minutes. The headspace N₂O was then transferred into avacuumed gas phase IR cell using a cannula, and was analyzed using aPerkin-Elmer FT-IR. A calibration curve was obtained by integration ofthe N₂O feature peaks at 2235 cm⁻¹ and 2212 cm⁻¹. These IR spectra ofN₂O standards are shown in FIG. 19A and the corresponding calibrationcurve is shown in FIG. 19B.

For the bulk electrolysis experiment, an airtight glass cell containing10 mL of different levels of nitrite (50, 100, 200 and 400 mM) in 2 mMCuTPMA, 0.2 M NaCl and 0.5 M MOPS buffer (pH 7.2) was used. Thesolutions were first purged with Ar for 30 minutes before eachexperiment. A Pt wire electrode (0.32 cm²) was used as the workingelectrode and Ag/AgCl wire was used as the reference/counter electrode.A constant potential (−0.4 V vs. 0.2 M Ag/AgCl) was applied for 3 hourswith stirring, after which the headspace gas was transferred into avacuumed gas phase IR cell using a cannula, and was analyzed using aPerkin-Elmer FT-IR. These results are shown in FIG. 19C. As depicted, asthe level of nitrite was increased, the level of N₂O that was produceddecreased dramatically.

As illustrated in these examples, nitric oxide gas can be efficientlygenerated from a source of nitrite ions 26 or a medium 27 (including theCu(II)-ligand complex) using electrodes and, respectively, the two-stepvoltage cycle or the application of a constant cathodic voltage. In someexamples, the reductive cleaning of the electrode prepares the electrodefor anodic liberation of Cu(I) ions, which react with nitrite togenerate NO. It is believed that the processes disclosed herein can beemployed to modulate the release of NO from polymer surfaces used toprepare, for example, in-dwelling catheters. Since continuous fluxes ofNO to kill or disperse biofilm forming on microbes or to preventplatelet induced clotting may not be necessary, it is believed thatcatheters and other medical devices disclosed herein may be utilizedeffectively for longer periods of time with a given supply of nitrite.This is due, at least in part, to the ability to turn the NO release onand off. It is further believed that when NO is produced at high enoughlevels in pulses at a defined frequency, the total moles of NO requiredfor delivery over a given time period will be much less than the totalmoles of NO required with continuous release NO donor chemistry.

It is to be understood that the ranges provided herein include thestated range and any value or sub-range within the stated range. Forexample, a range from about 1 cm to about 2 cm should be interpreted toinclude not only the explicitly recited limits of about 1 cm to about 2cm, but also to include individual values, such as 1.5 cm, 1.75 cm,etc., and sub-ranges, such as from about 1.25 cm to about 1.75 cm, etc.Furthermore, when “about” is utilized to describe a value, this is meantto encompass minor variations (up to +/−5%) from the stated value. Whenapplied potential values are discussed, it is to be understood thatwider ranges may be suitable. In some of the examples disclosed herein,increasing the magnitude of the anodic potential pulse increases theamount of Cu(I), and thus also increases the amount of NO generated. Inother of the examples disclosed herein, increasing the magnitude of thecathodic potential pulse (i.e., a more negative cathodic potential)increases the amount of Cu(I)-ligand complex that is generated, and thusalso increases the amount of NO generated. As such, it is believed thata broad range is applicable for the applied potential values, the limitsof which may depend on the desired amount of Cu(I) species and NO to begenerated, or on the time required to clean the working electrode.

Furthermore, reference throughout the specification to “one example”,“another example”, “an example”, and so forth, means that a particularelement (e.g., feature, structure, and/or characteristic) described inconnection with the example is included in at least one exampledescribed herein, and may or may not be present in other examples. Inaddition, it is to be understood that the described elements for anyexample may be combined in any suitable manner in the various examplesunless the context clearly dictates otherwise.

While several examples have been described in detail, it will beapparent to those skilled in the art that the disclosed examples may bemodified. Therefore, the foregoing description is to be considerednon-limiting.

What is claimed is:
 1. A nitric oxide delivery device, comprising: amedium including a source of nitrite ions and a Cu(II)-ligand complex; aworking electrode in contact with the medium; a reference/counterelectrode in contact with the medium and electrically isolated from theworking electrode; and a nitric oxide permeable material separating themedium from an external environment to contain blood.
 2. The nitricoxide delivery device as defined in claim 1 wherein the delivery deviceis a catheter having a lumen dedicated to housing the medium, theworking electrode, and the reference/counter electrode.
 3. The nitricoxide delivery device as defined in claim 2, further comprising a sealoperatively attached to the lumen.
 4. The nitric oxide delivery deviceas defined in claim 1 wherein the nitric oxide permeable material issilicone rubber.
 5. The nitric oxide delivery device as defined in claim1 wherein the medium is an aqueous solution including the source ofnitrite ions and the Cu(II)-ligand complex or a hydrogel including thesource of nitrite ions and the Cu(II)-ligand complex.
 6. The nitricoxide delivery device as defined in claim 1 wherein the Cu(II)-ligandcomplex is selected from the group consisting ofCu(II)-tri(2-pyridylmethyl)amine,Cu(II)-tri(2-dimethylamino)ethyl]amine,Cu(II)-tri(2-pyridylmethyl)phosphine, and combinations thereof.
 7. Thenitric oxide delivery device as defined in claim 1 wherein the source ofnitrite ions in the medium is any water soluble, inorganic nitrite salt.8. The nitric oxide delivery device as defined in claim 1, furthercomprising: a conductive lead electrically connected to the workingelectrode; and a second conductive lead electrically connected to thereference/counter electrode.
 9. The nitric oxide delivery device asdefined in claim 8 wherein: the conductive lead has a polymer coatingthereon; an other polymer coating is established i) on the secondconductive lead such that an end of the second conductive lead remainsexposed, and ii) on an area of the reference/counter electrode that isadjacent to the working electrode; and an other area of thereference/counter electrode remains exposed.
 10. The nitric oxidedelivery device as defined in claim 1 wherein the nitric oxide permeablematerial has a surface that is modified to detect surface charge or pH.11. The nitric oxide delivery device as defined in claim 10, furthercomprising an external reference electrode operatively positionedoutside of the nitrix oxide permeable material; and a high impedancevoltmeter operatively connected to the reference/counter electrode andthe external reference electrode to detect a surface charge change or apH change occurring at the modified surface.
 12. A gas delivery device,comprising: a nitric oxide generating system, including: a mediumincluding i) a source of nitrite ions, or ii) a source of nitrite ionsand a Cu(II)-ligand complex; a working electrode in contact with themedium, wherein i) when the medium includes the source of nitrite ions,the working electrode is a copper containing conductive material or abase material coated with a copper containing conductive material, orii) when the medium includes the source of nitrite ions and theCu(II)-ligand complex, the working electrode is selected from the groupconsisting of platinum, gold, carbon, a carbon coated material, andmercury; and a reference/counter electrode in contact with the mediumand electrically isolated from the working electrode; an inlet conduitto deliver oxygen gas to the medium; and an outlet conduit to transporta stream of oxygen gas and nitric oxide from the medium; and anoxygenator operatively connected to the outlet conduit, the oxygenatorincluding a membrane permeable to oxygen gas and nitric oxide, themembrane positioned to receive the stream of oxygen gas and nitric oxidefrom the outlet conduit at a first surface and to deliver a cleanedstream of oxygen gas and nitric oxide through a second surface.
 13. Thegas delivery device as defined in claim 12 wherein the oxygenator is ablood oxygenator that further includes: a housing; a plurality of themembranes positioned within the housing and operatively connected to theoutlet conduit, each of the membranes being a hollow polymeric fiberthat is permeable to oxygen gas and nitric oxide, wherein an interiorsurface of each hollow polymeric fiber is the first surface and anexterior surface of each hollow polymeric fiber is the second surface; ablood inlet to introduce blood into the housing adjacent to the secondsurfaces; and a blood exit to transport blood from the housing.
 14. Thegas delivery device as defined in claim 12 wherein the oxygenator is aninhalation unit that further includes a patient delivery system.
 15. Thegas delivery device as defined in claim 12 wherein: the medium includesthe source of nitrite ions; the medium is an aqueous solution or ahydrogel; and the source of nitrite ions is any water soluble, inorganicnitrite salt.
 16. The gas delivery device as defined in claim 12wherein: the medium includes the source of nitrite ions and theCu(II)-ligand complex; the medium is an aqueous solution or a hydrogel;the source of nitrite ions is any water soluble, inorganic nitrite salt;and the Cu(II)-ligand complex is selected from the group consisting ofCu(II)-tri(2-pyridylmethyl)amine,Cu(II)-tri(2-dimethylamino)ethyl]amine,Cu(II)-tri(2-pyridylmethyl)phosphine, and combinations thereof.
 17. Amethod for generating nitric oxide, comprising: applying a cathodicvoltage to a working electrode positioned in contact with a mediumincluding a source of nitrite ions and a Cu(II)-ligand complex, therebyreducing the Cu(II)-ligand complex to a Cu(I)-ligand complex whichreacts with nitrite from the source of nitrite ions to form the nitricoxide.
 18. The method as defined in claim 17, further comprisingmodulating a flux of the generated nitric oxide by any of altering anamount of a surface area of the working electrode that is exposed to themedium, or altering a concentration of any of the Cu(II)-ligand complexor the source of nitrite ions in the medium, or altering a magnitude ofthe cathodic voltage over time.
 19. The method as defined in claim 17wherein the applying of the cathodic voltage is accomplished atpredetermined intervals.
 20. The method as defined in claim 17 whereinthe applying of the cathodic voltage includes: applying a first cathodicvoltage to increase NO production for a predetermined time period; andthen applying a second cathodic voltage that is less negative than thefirst cathodic voltage to decrease NO production after the predeterminedtime period has expired.